Abstract
The calmodulin (CaM)-binding domain of isoform 4b of the plasma membrane Ca(2+) -ATPase (PMCA) pump is represented by peptide C28. CaM binds to either PMCA or C28 by a mechanism in which the primary anchor residue Trp-1093 binds to the C-terminal lobe of the extended CaM molecule, followed by collapse of CaM with the N-terminal lobe binding to the secondary anchor Phe-1110 (Juranic, N., Atanasova, E., Filoteo, A. G., Macura, S., Prendergast, F. G., Penniston, J. T., and Strehler, E. E. (2010) J. Biol. Chem. 285, 4015-4024). This is a relatively rapid reaction, with an apparent half-time of ~1 s. The dissociation of CaM from PMCA4b or C28 is much slower, with an overall half-time of ~10 min. Using targeted molecular dynamics, we now show that dissociation of Ca(2+)-CaM from C28 may occur by a pathway in which Trp-1093, although deeply embedded in a pocket in the C-terminal lobe of CaM, leaves first. The dissociation begins by relatively rapid release of Trp-1093, followed by very slow release of Phe-1110, removal of C28, and return of CaM to its conformation in the free state. Fluorescence measurements and molecular dynamics calculations concur in showing that this alternative path of release of the PMCA4b CaM-binding domain is quite different from that of binding. The intermediate of dissociation with exposed Trp-1093 has a long lifetime (minutes) and may keep the PMCA primed for activation.
Highlights
Plasma membrane Ca2ϩ -ATPase (PMCA) pumps are regulated by calmodulin (CaM) binding to the cytosolic tail
We show that dissociation of Ca2؉-CaM from C28 may occur by a pathway in which Trp-1093, deeply embedded in a pocket in the C-terminal lobe of CaM, leaves first
Association/Dissociation of PMCA4b and Calmodulin ments and molecular dynamics calculations based on the recently solved structure of the CaM-C28 complex [12], we show that the preferred pathway of dissociation may not be a simple reversal of association but instead involves rapid release of the N-terminal anchor Trp-1093 from the C-terminal lobe of CaM, followed by the much slower dissociation of the C-terminal anchor Phe-1110
Summary
Plasma membrane Ca2ϩ -ATPase (PMCA) pumps are regulated by calmodulin (CaM) binding to the cytosolic tail. Association/Dissociation of PMCA4b and Calmodulin ments and molecular dynamics calculations based on the recently solved structure of the CaM-C28 complex [12], we show that the preferred pathway of dissociation may not be a simple reversal of association but instead involves rapid release of the N-terminal anchor Trp-1093 from the C-terminal lobe of CaM, followed by the much slower dissociation of the C-terminal anchor Phe-1110. This biphasic dissociation mechanism allows for the existence of a stable intermediate complex in which CaM is bound only via its N-terminal lobe to the PMCA. The long half-life of this complex may keep PMCA4b “primed” for activation under conditions in which the free Ca2ϩ-CaM concentration is low
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